The Neuropeptide Y Y2 Receptor Is Coexpressed with Nppb in Primary Afferent Neurons and Y2 Activation Reduces Histaminergic and IL-31-Induced Itch.

Itch stimuli are detected by specialized primary afferents that convey the signal to the spinal cord, but how itch transmission is regulated is still not completely known. Here, we investigated the roles of the neuropeptide Y (NPY)/Y2 receptor system on scratch behavior. The inhibitory Y2 receptor is expressed on mouse primary afferents, and intrathecal administration of the Y2 agonist peptide YY (PYY)3-36 reduced scratch episode frequency and duration induced by compound 48/80, an effect that could be reversed by intrathecal preadministration of the Y2 antagonist BIIE0246. Also, scratch episode duration induced by histamine could be reduced by PYY3-36 In contrast, scratch behavior induced by α-methyl-5HT, protease-activated receptor-2-activating peptide SLIGRL, chloroquine, topical dust mite extract, or mechanical itch induced by von Frey filaments was unaffected by stimulation of Y2 Primary afferent neurons expressing the Npy2r gene were found to coexpress itch-associated markers such as natriuretic peptide precursor b, oncostatin M receptor, and interleukin (IL) 31 receptor A. Accordingly, intrathecal PYY3-36 reduced the scratch behavior induced by IL-31. Our findings imply that the NPY/Y2 system reduces histaminergic and IL-31-associated itch through presynaptic inhibition of a subpopulation of itch-associated primary afferents. SIGNIFICANCE STATEMENT: The spinal neuropeptide Y system dampens scratching behavior induced by histaminergic compounds and interleukin 31, a cytokine involved in atopic dermatitis, through interactions with the Y2 receptor. The Y2 receptor is expressed by primary afferent neurons that are rich in itch-associated neurotransmitters and receptors such as somatostatin, natriuretic peptide precursor b, and interleukin 31 receptors.

Comparison of cytotoxicities and anti-allergic effects of topical ocular dual-action anti-allergic agents.

BACKGROUND: To investigate the cytotoxicities of the topical ocular dual-action anti-allergic agents (alcaftadine 0.25%, bepotastine besilate 1.5%, and olopatadine HCL 0.1%) on human corneal epithelial cells (HCECs) and their anti-allergic effects on cultured conjunctival epithelial cells. METHODS: A Methylthiazolyltetrazolium(MTT)-based calorimetric assay was used to assess cytotoxicities using HCECs at concentrations of 10, 20 or 30% for exposure durations of 30 min, 1 h, 2 h, 12 h or 24 h. Cellular morphologies were evaluated by inverted phase-contrast and electron microscopy. Wound widths were measured 2 h, 18 h, or 24 h after confluent HCECs monolayers were scratched. Realtime PCR was used to quantify anti-allergic effects on cultured human conjunctival cells, in which allergic reactions were induced by treating them with Aspergillus antigen. RESULTS: Cell viabilities decreased in time- and concentration-dependent manners. Cells were detached from dishes and showed microvilli loss, cytoplasmic vacuoles, and nuclear condensation when exposed to antiallergic agents; alcaftadine was found to be least cytotoxic. Alcaftadine treated HCECs monolayers showed the best wound healing followed by bepotastine and olopatadine (p < 0.0001). All agents significantly reduced the gene expressions of allergic cytokines (IL-5, IL-25, eotaxin, thymus and activation-regulated chemokine, and thymic stromal lymphopoietin) and alcaftadine had the greatest effect (p < 0.0001 in all cases). CONCLUSIONS: Alcaftadine seems to have less side effects and better therapeutic effects than the other two anti-allergic agents tested. It may be more beneficial to use less toxic agents for patients with ocular surface risk factors or presumed symptoms of toxicity.

Tetrahydro-3-benzazepines with fluorinated side chains as NMDA and σ1 receptor antagonists: Synthesis, receptor affinity, selectivity and antiallodynic activity.

The class of tetrahydro-1H-3-benzazepines was systematically modified in 1-, 3- and 7-position. In particular, a F-atom was introduced in ß- or γ-position of the 4-phenylbutyl side chain in 3-position. Ligands with the F-atom in γ-position possess higher GluN2B affinity than analogs bearing the F-atom in ß-position. This effect was attributed to the reduced basicity of ß-fluoro amines. 3-Benzazepines with a benzylic OH moiety show moderate GluN2B affinity, but considerable selectivity over the σ2 receptor. However, removal of the benzylic OH moiety led to increased GluN2B affinity, but reduced GluN2B/σ2 selectivity. With respect to GluN2B affinity the phenol 17b with a γ-fluorophenylbutyl moiety in 3-position represents the most interesting fluorinated ligand (Ki(GluN2B) = 16 nM). Most of the synthesized ligands reveal either similar GluN2B and σ1 affinity or higher σ1 affinity than GluN2B affinity. The methyl ether 16b shows high σ1 affinity (Ki(σ1) = 6.6 nM) and high selectivity over a broad panel of receptors and transporters. The high antiallodynic activity in the mouse capsaicin assay proved the σ1 antagonistic activity of 16b.

Acute dopamine receptor blockade in substantia nigra pars reticulata: a possible model for drug-induced Parkinsonism.

Dopamine (DA) depletion modifies the firing pattern of neurons in the substantia nigra pars reticulata (SNr), shifting their mostly tonic firing toward irregularity and bursting, traits of pathological firing underlying rigidity and postural instability in Parkinson's disease (PD) patients and animal models of Parkinsonism (PS). Drug-induced Parkinsonism (DIP) represents 20-40% of clinical cases of PS, becoming a problem for differential diagnosis, and is still not well studied with physiological tools. It may co-occur with tardive dyskinesia. Here we use in vitro slice preparations including the SNr to observe drug-induced pathological firing by using drugs that most likely produce it, DA-receptor antagonists (SCH23390 plus sulpiride), to compare with firing patterns found in DA-depleted tissue. The hypothesis is that SNr firing would be similar under both conditions, a prerequisite to the proposal of a similar preparation to test other DIP-producing drugs. Firing was analyzed with three complementary metrics, showing similarities between DA depletion and acute DA-receptor blockade. Moreover, blockade of either nonselective cationic channels or Cav3 T-type calcium channels hyperpolarized the membrane and abolished bursting and irregular firing, silencing SNr neurons in both conditions. Therefore, currents generating firing in control conditions are in part responsible for pathological firing. Haloperidol, a DIP-producing drug, reproduced DA-receptor antagonist firing modifications. Since acute DA-receptor blockade induces SNr neuron firing similar to that found in the 6-hydroxydopamine model of PS, output basal ganglia neurons may play a role in generating DIP. Therefore, this study opens the way to test other DIP-producing drugs. NEW & NOTEWORTHY Dopamine (DA) depletion enhances substantia nigra pars reticulata (SNr) neuron bursting and irregular firing, hallmarks of Parkinsonism. Several drugs, including antipsychotics, antidepressants, and calcium channel antagonists, among others, produce drug-induced Parkinsonism. Here we show the first comparison between SNr neuron firing after DA depletion vs. firing found after acute blockade of DA receptors. It was found that firing in both conditions is similar, implying that pathological SNr neuron firing is also a physiological correlate of drug-induced Parkinsonism.

Metabolic Activation of Cholestatic Drug-Induced Bile Acid-Dependent Toxicity in Human Sandwich-Cultured Hepatocytes.

We previously reported a cell-based toxicity assay using sandwich-cultured hepatocytes in combination with a titrated amount of human bile acid (BA) species. In this assay, test compound-induced inhibition of BA efflux from sandwich-cultured hepatocytes leads to BA-dependent cell toxicity (BAtox, i.e., cell death due to the accumulation of BAs). Using this assay, we investigated whether 1-aminobenzotriazole (1-ABT; a nonselective cytochrome P450 inhibitor) enhanced or suppressed test compound-induced BAtox. There was a tendency that BAtox of many compounds was enhanced by 1-ABT in human hepatocytes; in contrast, such a tendency was not observed in rat hepatocytes. In particular, 1-ABT tended to enhance BAtox of several compounds (clopidogrel, ticlopidine, everolimus, etc.) in human, whereas 1-ABT tended to enhance BAtox of only ticlopidine in rat. These results indicate that this system can be used to evaluate BAtox while taking into account drug metabolism and the existence of an interspecies difference in the effect of 1-ABT treatment on BAtox.

Editor's Highlight: Candidate Risk Factors and Mechanisms for Tolvaptan-Induced Liver Injury Are Identified Using a Collaborative Cross Approach.

Clinical trials of tolvaptan showed it to be a promising candidate for the treatment of Autosomal Dominant Polycystic Kidney Disease (ADPKD) but also revealed potential for idiosyncratic drug-induced liver injury (DILI) in this patient population. To identify risk factors and mechanisms underlying tolvaptan DILI, 8 mice in each of 45 strains of the genetically diverse Collaborative Cross (CC) mouse population were treated with a single oral dose of either tolvaptan or vehicle. Significant elevations in plasma alanine aminotransferase (ALT) were observed in tolvaptan-treated animals in 3 of the 45 strains. Genetic mapping coupled with transcriptomic analysis in the liver was used to identify several candidate susceptibility genes including epoxide hydrolase 2, interferon regulatory factor 3, and mitochondrial fission factor. Gene pathway analysis revealed that oxidative stress and immune response pathways were activated in response to tolvaptan treatment across all strains, but genes involved in regulation of bile acid homeostasis were most associated with tolvaptan-induced elevations in ALT. Secretory leukocyte peptidase inhibitor (Slpi) mRNA was also induced in the susceptible strains and was associated with increased plasma levels of Slpi protein, suggesting a potential serum marker for DILI susceptibility. In summary, tolvaptan induced signs of oxidative stress, mitochondrial dysfunction, and innate immune response in all strains, but variation in bile acid homeostasis was most associated with susceptibility to the liver response. This CC study has indicated potential mechanisms underlying tolvaptan DILI and biomarkers of susceptibility that may be useful in managing the risk of DILI in ADPKD patients.

Application of a Mechanistic Model to Evaluate Putative Mechanisms of Tolvaptan Drug-Induced Liver Injury and Identify Patient Susceptibility Factors.

Tolvaptan is a selective vasopressin V2 receptor antagonist, approved in several countries for the treatment of hyponatremia and autosomal dominant polycystic kidney disease (ADPKD). No liver injury has been observed with tolvaptan treatment in healthy subjects and in non-ADPKD indications, but ADPKD clinical trials showed evidence of drug-induced liver injury (DILI). Although all DILI events resolved, additional monitoring in tolvaptan-treated ADPKD patients is required. In vitro assays identified alterations in bile acid disposition and inhibition of mitochondrial respiration as potential mechanisms underlying tolvaptan hepatotoxicity. This report details the application of DILIsym software to determine whether these mechanisms could account for the liver safety profile of tolvaptan observed in ADPKD clinical trials. DILIsym simulations included physiologically based pharmacokinetic estimates of hepatic exposure for tolvaptan and2 metabolites, and their effects on hepatocyte bile acid transporters and mitochondrial respiration. The frequency of predicted alanine aminotransferase (ALT) elevations, following simulated 90/30 mg split daily dosing, was 7.9% compared with clinical observations of 4.4% in ADPKD trials. Toxicity was multifactorial as inhibition of bile acid transporters and mitochondrial respiration contributed to the simulated DILI. Furthermore, simulation analysis identified both pre-treatment risk factors and on-treatment biomarkers predictive of simulated DILI. The simulations demonstrated that in vivo hepatic exposure to tolvaptan and the DM-4103 metabolite, combined with these 2 mechanisms of toxicity, were sufficient to account for the initiation of tolvaptan-mediated DILI. Identification of putative risk-factors and potential novel biomarkers provided insight for the development of mechanism-based tolvaptan risk-mitigation strategies.

Tolvaptan regulates aquaporin-2 and fecal water in cirrhotic rats with ascites.

AIM: To investigate the role of tolvaptan in regulating aquaporin (AQP)-2 expression and fecal water content in cirrhotic rats with ascites. METHODS: Cirrhosis with ascites was induced in rats by repetitive dorsal injection of CCl4 for 14 wk. In total, 84 cirrhotic rats with ascites divided into three groups (vehicle, 3 mg/kg and 5 mg/kg tolvaptan), and then further divided into five subgroups (days 1, 2, 3, 4, and 5). Blood samples were obtained to measure vasopressin and sodium concentrations. Rats were killed and colonic mucosa was scraped for analysis of protein expression and AQP-2 transcriptional level. The whole layer was fixed for hematoxylin&eosin (HE) staining and feces were collected for determination of fecal water content. CONCLUSION: Compared with vehicle, vasopressin decreased significantly in the tolvaptan groups from day 2 to a similar level in each treatment group. AQP-2 showed significant upregulation in cirrhotic rats with ascites compared with an untreated control group (100% ± 22.9% vs 22.2% ± 10.23%, P < 0.01). After administration of tolvaptan, AQP-2 expression began to decrease significantly from day 2 in each treatment group, but no significant difference was finally found between the treatment groups. Fecal water content in the distal colon was increased by 5 mg/kg tolvaptan on day 1 (66.8% ± 9.3% vs 41.4% ± 6.3%, in the vehicle group, P < 0.05). Fecal water content returned to baseline at day 4 at the latest in both treatment groups, and did not correspond to the change in AQP-2 expression. HE staining of the colonic mucosa showed no mucosal damage related to tolvaptan. CONCLUSION: Upregulation of AQP-2 in the distal colon is found in cirrhotic rats with ascites. Tolvaptan inhibits its expression and may decrease water reabsorption and induce diarrhea.

Inhibition of Human Hepatic Bile Acid Transporters by Tolvaptan and Metabolites: Contributing Factors to Drug-Induced Liver Injury?

Tolvaptan is a vasopressin V(2)-receptor antagonist that has shown promise in treating Autosomal Dominant Polycystic Kidney Disease (ADPKD). Tolvaptan was, however, associated with liver injury in some ADPKD patients. Inhibition of bile acid transporters may be contributing factors to drug-induced liver injury. In this study, the ability of tolvaptan and two metabolites, DM-4103 and DM-4107, to inhibit human hepatic transporters (NTCP, BSEP, MRP2, MRP3, and MRP4) and bile acid transport in sandwich-cultured human hepatocytes (SCHH) was explored. IC(50) values were determined for tolvaptan, DM-4103 and DM-4107 inhibition of NTCP (∼41.5, 16.3, and 95.6 µM, respectively), BSEP (31.6, 4.15, and 119 µM, respectively), MRP2 (>50, ∼51.0, and >200 µM, respectively), MRP3 (>50, ∼44.6, and 61.2 µM, respectively), and MRP4 (>50, 4.26, and 37.9 µM, respectively). At the therapeutic dose of tolvaptan (90 mg), DM-4103 exhibited a C(max)/IC(50) value >0.1 for NTCP, BSEP, MRP2, MRP3, and MRP4. Tolvaptan accumulation in SCHH was extensive and not sodium-dependent; intracellular concentrations were ∼500 µM after a 10-min incubation duration with tolvaptan (15 µM). The biliary clearance of taurocholic acid (TCA) decreased by 43% when SCHH were co-incubated with tolvaptan (15 µM) and TCA (2.5 µM). When tolvaptan (15 µM) was co-incubated with 2.5 µM of chenodeoxycholic acid, taurochenodeoxycholic acid, or glycochenodeoxycholic acid in separate studies, the cellular accumulation of these bile acids increased by 1.30-, 1.68-, and 2.16-fold, respectively. Based on these data, inhibition of hepatic bile acid transport may be one of the biological mechanisms underlying tolvaptan-associated liver injury in patients with ADPKD.

Human Sulfotransferases Enhance the Cytotoxicity of Tolvaptan.

Tolvaptan, a vasopressin receptor 2 antagonist used to treat hyponatremia, has recently been reported to be associated with liver injury. Sulfotransferases (SULTs) have been implicated as important detoxifying and/or activating enzymes for numerous xenobiotics, drugs, and endogenous compounds. To characterize better the role of SULTs in tolvaptan metabolism, HEK293 cells stably overexpressing 12 human SULTs were generated. Using these cell lines, the extent of tolvaptan sulfate formation was assessed by reversed-phase high-performance liquid chromatography through comparison to a synthetic standard. Of the 12 known human SULTs, no detectable sulfation of tolvaptan was observed with SULT1A1, SULT1A2, SULT1A3, SULT1C2, SULT1C4, SULT4A1, or SULT6B1. The affinity of individual SULT isozymes, as determined by Km analysis, was SULT1C3 >> SULT2A1 > SULT2B1 ∼ SULT1B1 > SULT1E1. The half inhibitory concentration of tolvaptan on cell growth in HEK293/SULT1C3 cells and HEK293/CYP3A4 & SULT1C3 cells was significantly lower than that in the corresponding HEK293/vector cells or HEK293/CYP3A4 & SULT vector cells. Moreover, exposing cells to tolvaptan in the presence of cyclosporine A, an inhibitor of the drug efflux transporters, significantly increased the intracellular levels of tolvaptan sulfate and decreased the cell viability in HEK293/SULT1C3 cells. These data indicate that sulfation increased the cytotoxicity of tolvaptan.

The glucagon-like peptide 1 (GLP-1) receptor agonist exendin-4 reduces cocaine self-administration in mice.

Glucagon-like peptide 1 (GLP-1) analogues are used for the treatment of type 2 diabetes. The ability of the GLP-1 system to decrease food intake in rodents has been well described and parallels results from clinical trials. GLP-1 receptors are expressed in the brain, including within the ventral tegmental area (VTA) and the nucleus accumbens (NAc). Dopaminergic neurons in the VTA project to the NAc, and these neurons play a pivotal role in the rewarding effects of drugs of abuse. Based on the anatomical distribution of GLP-1 receptors in the brain and the well-established effects of GLP-1 on food reward, we decided to investigate the effect of the GLP-1 analogue exendin-4 on cocaine- and dopamine D1-receptor agonist-induced hyperlocomotion, on acute and chronic cocaine self-administration, on cocaine-induced striatal dopamine release in mice and on cocaine-induced c-fos activation. Here, we report that GLP-1 receptor stimulation reduces acute and chronic cocaine self-administration and attenuates cocaine-induced hyperlocomotion. In addition, we show that peripheral administration of exendin-4 reduces cocaine-induced elevation of striatal dopamine levels and striatal c-fos expression implicating central GLP-1 receptors in these responses. The present results demonstrate that the GLP-1 system modulates cocaine's effects on behavior and dopamine homeostasis, indicating that the GLP-1 receptor may be a novel target for the pharmacological treatment of drug addiction.

Mechanisms of tolvaptan-induced toxicity in HepG2 cells.

Tolvaptan, a vasopressin receptor 2 antagonist used to treat hyponatremia, has recently been reported to be associated with an increased risk of liver injury. In this study, we explored the underlying mechanisms of hepatotoxicity of tolvaptan using human HepG2 cells. Tolvaptan inhibited cell growth and caused cell death in a concentration- and time-dependent manner. Tolvaptan treatment led to delayed cell cycle progression, accompanied by decreased levels of several cyclins and cyclin-dependent kinases. Tolvaptan was found to cause DNA damage, as assessed by alkaline comet assays; this was confirmed by increased levels of 8-oxoguanine and phosphorylation of histone H2AX. Exposure of HepG2 cells to tolvaptan enhanced cytochrome C release and triggered apoptosis by modulating Bcl-2 family members. The activation of p38 contributed to tolvaptan-mediated apoptosis via down-regulation of Bcl-2. Proteasome inhibition altered tolvaptan-induced cell cycle deregulation and enhanced tolvaptan-induced apoptosis and cytotoxicity. Moreover, tolvaptan treatment induced autophagy. Inhibition of autophagy by knocking-down an autophagy-related gene increased tolvaptan-induced apoptosis and cytotoxicity. Taken together, our findings suggest that the cytotoxicity of tolvaptan results from delayed cell cycle progression, the induction of DNA damage, and the execution of apoptosis. In addition, a number of signaling pathways were perturbed by tolvaptan and played an important role in its cytotoxicity.

Combination effects of AHR agonists and Wnt/ß-catenin modulators in zebrafish embryos: Implications for physiological and toxicological AHR functions.

Wnt/ß-catenin signaling regulates essential biological functions and acts in developmental toxicity of some chemicals. The aryl hydrocarbon receptor (AHR) is well-known to mediate developmental toxicity of persistent dioxin-like compounds (DLCs). Recent studies indicate a crosstalk between ß-catenin and the AHR in some tissues. However the nature of this crosstalk in embryos is poorly known. We observed that zebrafish embryos exposed to the ß-catenin inhibitor XAV939 display effects phenocopying those of the dioxin-like 3,3',4,4',5-pentachlorobiphenyl (PCB126). This led us to investigate the AHR interaction with ß-catenin during development and ask whether developmental toxicity of DLCs involves antagonism of ß-catenin signaling. We examined phenotypes and transcriptional responses in zebrafish embryos exposed to XAV939 or to a ß-catenin activator, 1-azakenpaullone, alone or with AHR agonists, either PCB126 or 6-formylindolo[3,2-b]carbazole (FICZ). Alone 1-azakenpaullone and XAV939 both were embryo-toxic, and we found that in the presence of FICZ, the toxicity of 1-azakenpaullone decreased while the toxicity of XAV939 increased. This rescue of 1-azakenpaullone effects occurred in the time window of Ahr2-mediated toxicity and was reversed by morpholino-oligonucleotide knockdown of Ahr2. Regarding PCB126, addition of either 1-azakenpaullone or XAV939 led to lower mortality than with PCB126 alone but surviving embryos showed severe edemas. 1-Azakenpaullone induced transcription of ß-catenin-associated genes, while PCB126 and FICZ blocked this induction. The data indicate a stage-dependent antagonism of ß-catenin by Ahr2 in zebrafish embryos. We propose that the AHR has a physiological role in regulating ß-catenin during development, and that this is one point of intersection linking toxicological and physiological AHR-governed processes.

9- and 11-Substituted 4-azapaullones are potent and selective inhibitors of African trypanosoma.

Trypanosomes from the "brucei" complex are pathogenic parasites endemic in sub-Saharan Africa and causative agents of severe diseases in humans and livestock. In order to identify new antitrypanosomal chemotypes against African trypanosomes, 4-azapaullones carrying α,ß-unsaturated carbonyl chains in 9- or 11-position were synthesized employing a procedure with a Heck reaction as key step. Among the so prepared compounds, 5a and 5e proved to be potent antiparasitic agents with antitrypanosomal activity in the submicromolar range.

Single dose varenicline may trigger epileptic activity.

Varenicline is a new drug for smoking cessation, and its effect on epilepsy is not clear. The aim of this study was to investigate whether different doses of varenicline cause epileptic activity. Forty rats were randomly assigned to the following eight groups: control, saline, and 0.025, 0.04, 0.1, 0.5, 1, and 2 mg kg(-1) varenicline (single dose, i.p.). EEGs were recorded before the varenicline injection and during the following 240 min. While epileptic discharges were observed on the EEGs of the rats in all of the varenicline-treated groups, motor findings of epileptic seizure were not observed in some rats in these groups except the 1 and 2 mg kg(-1) groups. These findings indicate that different single doses of varenicline cause epileptic activity in rats.

A phase I dose-finding study of the novel Toll-like receptor 8 agonist VTX-2337 in adult subjects with advanced solid tumors or lymphoma.

PURPOSE: This phase I, open-label, uncontrolled, ascending-dose study explored the safety, maximum tolerated dose (MTD), pharmacokinetics, and pharmacology of the TLR8 agonist VTX-2337 in subjects with advanced solid tumors or lymphoma. EXPERIMENTAL DESIGN: VTX-2337 doses (0.1-3.9 mg/m(2)) were administered subcutaneously on days 1, 8, and 15 of each 28-day cycle. Safety/tolerability assessments included adverse events (AE); physical, ophthalmologic, and laboratory evaluations; and electrocardiograms. Dose-limiting toxicities (DLT) were evaluated during the first cycle. Pharmacokinetics were evaluated after the first dose. Plasma samples were quantitatively assessed for chemokines, cytokines, and other inflammatory mediators. Antitumor activity was assessed. RESULTS: Thirty-three subjects were enrolled in 8 cohorts and received an average of 2 treatment cycles (range, 1-8 cycles). Most AEs were grades 1 to 2; the most common drug-related AEs were injection site reactions, chills, pyrexia, and influenza-like illness. One DLT was reported: grade 3 hypotension (3.9 mg/m(2)). The MTD was considered the highest dose administered. Peak drug plasma levels and total systemic exposure were generally dose proportional. At doses ≥0.4 mg/m(2), increases above baseline levels were observed for plasma levels of G-CSF, monocyte chemoattractant protein-1, macrophage inflammatory protein-1ß, and TNFα. Eight subjects (24.2%) had a best response of stable disease (median duration, 54.5 days). CONCLUSIONS: VTX-2337 is clinically well tolerated and biologically active with a predictable pharmacokinetic profile. Suitable doses for testing in combination studies were identified. Phase II placebo-controlled studies of VTX-2337 in combination with doxorubicin in ovarian cancer, and in combination with platinum chemotherapy, 5 FU, and cetuximab in head and neck cancer have been initiated (NCT #01666444 and NCT#01836029).

Case report of ivabradine intoxication.

Ivabradine is a drug used for the treatment of angina and chronic heart failure in cases of intolerance or insufficiency of response to beta-blocker treatment. A 47-year-old man was admitted to the emergency department of the hospital for a voluntary intoxication with 280 mg of ivabradine: he presented drowsiness and a mild sinusal bradycardia (50 bpm) associated with a well-tolerated low blood pressure at 100/50 mmHg. No complication was noted and he was discharged from the hospital on Day 3. A method for ivabradine assay in serum was obtained using liquid chromatography coupled with a mass spectrometry detection method. After a deproteinization step using QuECHERS salts and acetonitrile, a chromatographic separation was performed using a 5-µm 50 × 2.1 mm Xterra® column (Waters, France). Detection was performed using an LTQ linear ion-trap mass spectrometer equipped with an electrospray ionization source used in a positive ionization mode (ThermoFisher Scientific, San Jose CA, USA) and a detection in full MS(2) scan. The limit of quantification of ivabradine was 10 µg/L, and the method was linear up to 1000 µg/L. The ivabradine concentration in the patient's serum was 375 µg/L. This concentration value was >30 times those measured after therapeutic doses intakes. Nevertheless, the bradycardia was no more severe than the one observed with therapeutic dosage. In conclusion, this case tends to show an absence of correlation between blood concentration and severity of the troubles in cases of overdosage.

Minocycline prevents osmotic demyelination associated with aquaresis.

Overly rapid correction of chronic hyponatremia can cause osmotic demyelination syndrome (ODS). Minocycline protects ODS associated with overly rapid correction of chronic hyponatremia with hypertonic saline infusion in rats. In clinical practice, inadvertent rapid correction frequently occurs due to water diuresis, when vasopressin action suddenly ceases. In addition, vasopressin receptor antagonists have been applied to treat hyponatremia. Here the susceptibility to and pathology of ODS were evaluated using rat models developed to represent rapid correction of chronic hyponatremia in the clinical setting. The protective effect of minocycline against ODS was assessed. Chronic hyponatremia was rapidly corrected by 1 (T1) or 10 mg/kg (T10) of tolvaptan, removal of desmopressin infusion pumps (RP), or administration of hypertonic saline. The severity of neurological impairment in the T1 group was significantly milder than in other groups and brain hemorrhage was found only in the T10 and desmopressin infusion removal groups. Minocycline inhibited demyelination in the T1 group. Further, immunohistochemistry showed loss of aquaporin-4 (AQP4) in astrocytes before demyelination developed. Interestingly, serum AQP4 levels were associated with neurological impairments. Thus, minocycline can prevent ODS caused by overly rapid correction of hyponatremia due to water diuresis associated with vasopressin action suppression. Increased serum AQP4 levels may be a predictive marker for ODS.

In vivo genotoxicity of a novel heterocyclic amine, aminobenzoazepinoquinolinone-derivative (ABAQ), produced by the Maillard reaction between glucose and l-tryptophan.

We recently demonstrated that a novel heterocyclic amine, 5-amino-6-hydroxy-8H-benzo[6,7]azepino[5,4,3-de]quinolin-7-one (ABAQ), is produced from glucose and l-tryptophan by the Maillard reaction at physiological temperature and pH, and that ABAQ was strongly mutagenic for Salmonella strains in the presence of S9 mix. Here, we present the results of three in vivo genotoxicity assays of ABAQ. The comet assay revealed that DNA damage was significantly increased in the livers, kidneys, lungs, and bone marrows of ICR mice, 3h after i.p. injection of ABAQ (50mg/kg body weight (bw)). To evaluate clastogenicity, the peripheral blood micronucleus test was performed, also in ICR mice. ABAQ induced micronucleated reticulocytes (MNRETs) in a dose-dependent manner; the frequency of MNRETs was significantly elevated at all i.p. doses (12.5, 25, and 50mg/kg bw) after 48h. To investigate the mutagenicity of ABAQ in vivo, gpt delta transgenic mice were treated with five consecutive administrations of ABAQ by gavage at doses of 25 or 50mg/kg per week for 3 weeks. The frequencies of gpt mutations (MF) in the liver of mice increased significantly compared with controls, in a dose-dependent manner. No significant increase of gpt MF was detected in the kidneys. Base substitutions predominated; both G:C→A:T and A:T→C:G mutations were significantly increased by ABAQ. The Spi(-) MF was also significantly increased in the liver after ABAQ treatment. If formed in vivo, ABAQ may give rise to adverse genotoxic effects.

Comparative pathophysiology, toxicology, and human cancer risk assessment of pharmaceutical-induced hibernoma.

In humans, hibernoma is a very rare, benign neoplasm of brown adipose tissue (BAT) that typically occurs at subcutaneous locations and is successfully treated by surgical excision. No single cause has been accepted to explain these very rare human tumors. In contrast, spontaneous hibernoma in rats is rare, often malignant, usually occurs in the thoracic or abdominal cavity, and metastases are common. In recent years, there has been an increased incidence of spontaneous hibernomas in rat carcinogenicity studies, but overall the occurrence remains relatively low and highly variable across studies. There have only been four reported examples of pharmaceutical-induced hibernoma in rat carcinogenicity studies. These include phentolamine, an alpha-adrenergic antagonist; varenicline, a nicotine partial agonist; tofacitinib, a Janus kinase (JAK) inhibitor; and hydromorphone, an opiod analgesic. Potential non-genotoxic mechanisms that may contribute to the pathogenesis of BAT activation/proliferation and/or subsequent hibernoma development in rats include: (1) physiological stimuli, (2) sympathetic stimulation, (3) peroxisome proliferator-activated receptor (PPAR) agonism, and/or (4) interference or inhibition of JAK/Signal Transducer and Activator of Transcription (JAK/STAT) signaling. The evaluation of an apparent increase of hibernoma in rats from 2-year carcinogenicity studies of novel pharmaceutical therapeutics and its relevance to human safety risk assessment is complex. One should consider: the genotoxicity of the test article, dose/exposure and safety margins, and pathophysiologic and morphologic differences and similarities of hibernoma between rats and humans. Hibernomas observed to date in carcinogenicity studies of pharmaceutical agents do not appear to be relevant for human risk at therapeutic dosages.